Cfx96 system

For RNA extraction, total RNAs were isolated using Trizol reagent (Invitrogen) and 1 µg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen). The qRT-PCR was conducted using a Bio-Rad CFX96 system with SYBR green to determine the mRNA expression level of a gene of interest. Expression levels were normalized to the expression of TATA box binding protein TBP RNA. Primers used for genes of interest were listed in Supplementary Table S1. The miRNAs were also reversed transcribed from total RNA. Briefly, 1 µg of total RNA was processed for poly A addition by adding 2 units of polymerase with 1-mM ATP in 1x RT buffer at 37 °C for 20 min in 10-μl volume, then adding 50-pmol anchor primer to 11 μl, incubating at 65 °C for 5 min, then 4 °C for 2 min. For the last step of cDNA synthesis, we added 2-µl 5x RT buffer, 2 µl 10-mM dNTP, 1-µl reverse transcriptase to total 20 µl, and incubated at 42 °C for 1 h. qRT-PCR was conducted using a Bio-Rad CFX96 system with Tagman probe to determine the miRNA expressions. Expression levels were normalized to the expression of 5s RNA and/or U6.

Total RNA, including miRNAs, from FFPE tissue was isolated using the miRNeasy FFPE kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions, including treatment with deparaffinization solution (Qiagen, Hilden, Germany).
For miR-195-5p detection, total RNA was reverse transcribed using a TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Real-time RT-PCR for miR-195-5p quantification was performed in 20 μL of final volume using the CFX96 System (Biorad Laboratories, Hercules, CA, USA) with TaqMan Advanced miRNA assays and TaqMan Fast Advanced Master mix (Thermo Fisher Scientific, Waltham MA, USA). miR-26a-5p and miR-186-5p were used as endogenous controls to perform normalization for human and mouse data, respectively.
For JUP analysis, total RNA was retrotranscribed using SuperScript™ VILO™ MasterMix (Thermo Fisher Scientific, Bremen, Germany). Quantitative real-time PCR was performed on a CFX96 System (Biorad Laboratories, Hercules, CA, USA) using the SsoAdvanced Universal SYBR Green Supermix (BioRad Laboratories, Hercules, CA, USA) and the QuantiTect Primer Assay for JUP and GAPDH (Qiagen, Hilden, Germany). GAPDH gene amplification was used as reference standard to normalize the relative expression of JUP.
The relative expression was calculated using the 2^−ΔCt and 2^−ΔΔCt formulas.

Total RNAs were isolated using Trizol reagent (Thermo Fisher Scientific), and 1 µg of total RNA subjected to reverse transcription using Superscript III transcriptase Thermo Fisher Scientific. Quantitative real-time PCR analysis was conducted using the Bio-Rad CFX96 system with SYBR green to determine mRNA expression levels of a gene of interest, which were normalized to expression of GAPDH. The miRNAs were isolated using PureLink® miRNA kit. Briefly, 50 ng of small RNAs were processed for poly A addition by adding 1 unit of polymerase with 1 mM ATP in 1 × RT buffer at 37 °C for 10 min in 10-μl volume, heat-inactivated at 95 °C for 2 min, then added 50 pmol anchor primer to 12.5 μl, and incubated at 65 °C for 5 min. For the last step of cDNA synthesis, we added 2 μl of 5× RT buffer, 2 μl of 10 mM dNTP, and 1 μl of reverse transcriptase to a total of 20 μl, and incubated at 42 °C for 1 h. Quantitative real-time PCR (QPCR) was conducted using the Bio-Rad CFX96 system with Taqman probe to determine the miRNA expression levels, which were normalized to the expression of 5S and/or U6.

The relative mRNA expression of TSSK1B in each tissue sample of yak and cattle–yak was detected by RT-qPCR, following the previous study [20 (link)]. The primers of TSSK1B for RT-qPCR were designed, as shown in Table 1. Each amplification system (20 μL) included 1 μL of forward and reverse primers, 10 μL of 2×NovoStart^®SYBR qPCR SuperMix Plus (Novoprotein, Suzhou, China), and 1 μL of diluted cDNA supplied with 7 μL RNase free water up to 20 μL. The CFX96^TM system (Bio-Rad, Hercules, CA, USA) was used with the following program steps: 95 °C for 1 min, 40 cycles of 95 °C for 20 s, and 60 °C for 1 min, followed by dissociation curve and cool down. The relative fold change of genes was calculated by 2^–∆∆Ct method, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as the housekeeping gene for data normalization. Each sample analysis was repeated independently in triplicate. For easy comparison of the relative expression of TSSK1B in various organs, the expression in heart was set to 1 (control group). The TSSK1B expression of adult yak testis was also set as the control group when contrasting the differential expression in yak testes of different ages and the expression in adult cattle–yak testes.

Three switchgrass seedlings from each pots were selected, washed, chopped, and mixed, and the samples were then weighed. Total RNA was extracted by using TRIzol^® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The quality of the extracted total RNA was checked by Nanodrop ND-1000 and gel electrophoresis analyses. Reverse transcription (RT) reactions were implemented with a One Step PrimeScript^® miRNA cDNA synthesis kit (TaKaRa, Dalian, China). qRT-PCR was run on a BIO-RAD CFX96TM system, and three biological replicates were performed for each miRNA. The qRT-PCR results were analysed by using the 2^−ΔΔCT method [37 (link)].

The total RNA of samples was extracted from plant seedlings using Trizol solution (Vazyme Biotech Co., Ltd., Nanjing, China). The complementary DNA (cDNA) templates were synthesized using the HiScriptII first strand cDNA synthesis kit (Vazyme Biotech Co., Ltd., Nanjing, China) [58 (link)]. Using SYBR PremixEx-Taq^TMII (Takara Bio Inc., Kusatsu, Japan), the RNA transcripts of the relative genes were measured using a real-time CFX96TM system (Bio-Rad, Hercules, CA, USA) for qRT-PCR [51 ]. The following procedure was performed for qRT-PCR: 94 ◦C for 3 min; 39 cycles of denaturation at 94 ◦C for 10 s, annealing at 57 ◦C for 10 s, elongation at 72 ◦C for 30 s. The reference gene was Actin-3 in soybean. Finally, the relative expression values were calculated using the comparative cycle threshold method 2^−△△ct. The experiments were carried out with three independent organisms [25 (link)].